Total Internal Reflection Fluorescence Microscopy

Total Internal Reflection Fluorescence (TIRF) Microscopy imaging was conducted as previously described (Loerke et al., 2009 (link)). Cells were grown on a gelatin-coated 22 × 22 mm glass (Corning, #2850–22) overnight and then mounted to a 25 × 75 mm cover slide (Thermo Scientific, #3050). Imaging was conducted with a 60X, 1.49-NA Apo TIRF objective (Nikon) mounted on a Ti-Eclipse inverted microscope equipped with an additional 1.8X tube lens, yielding a final magnification of 108X. Perfect focus was applied during time-lapsed imaging. For EPI-TIRF imaging, nearly simultaneous two channel (488 epifluorescence/TIRF) movies were acquired with multi-dimension acquisition (MDA). Movies were acquired at the rate of 1 frame/s. cmeAnalysis was applied for CCP detection and tracking (Aguet et al., 2013 (link); Jaqaman et al., 2008 (link); Loerke et al., 2011 (link)). Variation could arise from the heterogeneity of cover glass by itself and the gelatin-coating.